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OBJECTIVE: Genome-scale CRISPR-Cas9 knockout coupled with single-cell RNA sequencing (scRNA-seq) has been used to identify function-related genes. However, this method may knock out too many genes, leading to low efficiency in finding genes of interest. Insulin secretion is controlled by several electrophysiological events, including fluxes of KATP depolarization and K+ repolarization. It is well known that glucose stimulates insulin secretion from pancreatic ß-cells, mainly via the KATP depolarization channel, but whether other nutrients directly regulate the repolarization K+ channel to promote insulin secretion is unknown. METHODS: We used a system involving CRISPR-Cas9-mediated knockout of all 83 K+ channels and scRNA-seq in a pancreatic ß cell line to identify genes associated with insulin secretion. RESULTS: The expression levels of insulin genes were significantly increased after all-K+ channel knockout. Furthermore, Kcnb1 and Kcnh6 were the two most important repolarization K+ channels for the increase in high-glucose-dependent insulin secretion that occurred upon application of specific inhibitors of the channels. Kcnh6 currents, but not Kcnb1 currents, were reduced by one of the amino acids, lysine, in both transfected cells, primary cells and mice with ß-cell-specific deletion of Kcnh6. CONCLUSIONS: Our function-related CRISPR screen with scRNA-seq identifies Kcnh6 as a lysine-specific channel.
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Insulina , Lisina , Camundongos , Animais , Secreção de Insulina , Lisina/metabolismo , Insulina/metabolismo , Glucose/farmacologia , Trifosfato de Adenosina/metabolismoRESUMO
Alzheimer's disease (AD) is one of the leading causes of death and disability. AD is a devastating disease that has caused an overwhelming burden. However, no disease-modified treatment was discovered. The approval of sodium oligomannate (GV-971) in mild-moderate AD patients has attracted great attention to investigate the role of saccharides in AD. Therefore, summarizing and explaining the role of saccharides in AD is urgent and promising. Recent studies showed that polysaccharides (PSs) potentially benefit AD in vitro and in vivo. PSs could alleviate the pathological damage and improve cognitive symptoms via (1) antagonizing the toxicity of abnormal amyloid-beta and tau proteins; (2) attenuating oxidative stress and proinflammation; (3) rebuilding neuroplasticity. PSs exhibit one-multiple pathological hits of AD. However, a thorough chemical investigation is needed for further study.
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PURPOSE: To investigate whether resveratrol promotes odontogenic differentiation of human dental pulp stem cells(DPSCs) by up-regulating the expression of silent information regulator 1 (SIRT1) and activating ß-catenin signaling pathway. METHODS: Different concentrations of resveratrol(0, 10, 15, 20 and 50 µmol/L) were used to treat DPSCs for 7 days and 14 days, and cell proliferative activity was detected by CCK-8. After odontogenic differentiation induced by 15 µmol/L resveratrol for 7 days, alkaline phosphatase(ALP) staining was performed and real-time quantitative reverse transcription PCR(qRT-PCR) was used to detect the mRNA expression of Runt-related transcription factor 2 (Runx2), dentin sialophosphoprotein(DSPP) and dentin matrix protein-1(DMP-1) in DPSCs. Western blot was used to detect the expression of SIRT1 in DPSCs on a specific day (0, 3rd, 5th, 7th and 14th) after differentiation induction. Western blot was also used to detect the expression of SIRT1 and activated ß-catenin during odontogenic differentiation of DPSCs treated by 15 µmol/L resveratrol for 7 days. The experimental data was analyzed with GraphPad Prism 9 software package. RESULTS: 15 µmol/L resveratrol had no significant effect on proliferation of DPSCs on the 7th and 14th day; 15 µmol/L resveratrol promoted odontogenic differentiation of DPSCs and up-regulated mRNA expression of RUNX2, DSPP, and DMP-1 in DPSCs; the expression of SIRT1 was the highest on the 7th day during odontogenic differentiation induction. Resveratrol resulted in the increasing protein expressions of SIRT1 and activated ß-catenin when DPSCs was induced to odontogenic differentiation for 7 days. CONCLUSIONS: Resveratrol promotes odontogenic differentiation of human DPSCs by up-regulating the expression of SIRT1 protein and activating ß-catenin signaling pathway.
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Subunidade alfa 1 de Fator de Ligação ao Core , beta Catenina , Humanos , Resveratrol/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , beta Catenina/metabolismo , beta Catenina/farmacologia , Polpa Dentária/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Sirtuína 1/farmacologia , Proliferação de Células , Diferenciação Celular , Odontogênese/genética , Células-Tronco/metabolismo , RNA Mensageiro/metabolismo , Células CultivadasRESUMO
PURPOSE: To investigate the effects of chitosan oligosaccharide on bone metabolism and IKK/NF-κB pathway in mice with osteoporosis and periodontitis. METHODS: Thirty rats were randomly divided into 3 groups, with 10 rats in each group. They were divided into control group, ovariectomized periodontitis group and chitosan oligosaccharide treatment group. Except for the control group, the other two groups were ovariectomized and smeared with Porphyromonas gingivalis fluid to establish the model of osteoporosis with periodontitis. Four weeks after ligation, the rats in chitosan oligosaccharide treatment group were gavaged with 200 mg/kg chitosan oligosaccharide, and the other two groups were gavaged with equal volume of normal saline once a day for 90 days. The periodontal tissues of each group were observed before administration, and the bone mineral density of rats was detected by dual energy X-ray animal bone mineral density and body composition analysis system. After 90 days of administration, the bone mineral density was detected again. After administration, blood was collected from tail vein, and the contents of serum alkaline phosphatase (ALP), bone Gla protein (BGP) and tartrate resistant acid phosphatase 5b (TRACP5b) were measured by enzyme-linked immunodeficient assay. The gingival index and periodontal attachment loss of rats in each group were obtained by visual examination and exploratory examination. The maxilla was removed, and the distance from the enamel cementum boundary to the alveolar crest was measured to obtain alveolar bone absorption value. H-E staining was used to observe the pathology of maxilla in each group. RT-PCR and Western blot were used to detect the nuclear factors in periodontal tissue of rats in each group. SPSS 22.0 software package was used for statistical analysis. RESULTS: Before administration, the gums of the control group were pink without bleeding, and the gums of the other two groups were red and swollen with slight bleeding. After administration, compared with the control group, the bone mineral density, serum ALP, BGP of ovariectomized periodontitis group decreased significantly(Pï¼0.05); while TRACP5b, gingival index, loss of periodontal attachment and alveolar bone resorption, NF-κB and IKK mRNA and protein expression in periodontal tissue increased significantly(Pï¼0.05). Compared with the ovariectomized periodontitis group, the bone mineral density, serum ALP, BGP were significantly increased(Pï¼0.05); while TRACP5b, gingival index, periodontal attachment loss and alveolar bone resorption, NF-κB and IKK mRNA and protein expression in periodontal tissue were significantly decreased (Pï¼0.05). In the ovariectomized periodontitis group, the periodontal tissue combined with epithelium was separated from the tooth surface, the dental pocket was obvious and deep, and the height of alveolar bone decreased. Although dental pocket could be observed in the periodontal tissue of rats treated with chitosan oligosaccharide, it was not obvious, and new bone appeared around the alveolar bone. CONCLUSIONS: Chitosan oligosaccharide can induce biochemical indexes of bone metabolism to become normal, alleviate the symptoms of periodontitis, this may be related to the inhibition of IKK/NF-κB pathway by chitosan oligosaccharide.
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Perda do Osso Alveolar , Quitosana , Osteoporose , Periodontite , Ratos , Camundongos , Animais , NF-kappa B , Perda da Inserção Periodontal , Osteoporose/tratamento farmacológico , Periodontite/tratamento farmacológico , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/prevenção & controle , Oligossacarídeos/farmacologiaRESUMO
Pseudolaric acid B (PAB), a natural product isolated from the root bark of Pseudolarix kaempferi, has been reported to exert inhibitory effects in various cancers. However, the underlying mechanisms remain largely unclear. In the present study, we investigated the mechanism through which PAB exert its anticancer effects in hepatocellular carcinoma (HCC). PAB inhibited the viability of and induced apoptosis in Hepa1-6 cells in a dose-dependent manner. It disrupted mitochondrial membrane potential (MMP) and impaired ATP production. Furthermore, PAB induced phosphorylation of DRP1 at Ser616 and mitochondrial fission. Blocking DRP1 phosphorylation by Mdivi-1 inhibited mitochondrial fission and PAB-induced apoptosis. Moreover, c-Jun N-terminal kinase (JNK) was activated by PAB, and blocking JNK activity using SP600125 inhibited PAB-induced mitochondrial fission and cell apoptosis. Furthermore, PAB activated AMP-activated protein kinase (AMPK), and inhibiting AMPK by compound C attenuated PAB-stimulated JNK activation and blocked DRP1-dependent mitochondrial fission and apoptosis. Our in vivo data confirmed that PAB inhibited tumor growth and induced apoptosis in an HCC syngeneic mouse model by inducing the AMPK/JNK/DRP1/mitochondrial fission signaling pathway. Furthermore, a combination of PAB and sorafenib showed a synergistic effect in inhibiting tumor growth in vivo. Taken together, our findings highlight a potential therapeutic strategy for HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Camundongos , Carcinoma Hepatocelular/tratamento farmacológico , Proteínas Quinases Ativadas por AMP , Dinâmica Mitocondrial , Neoplasias Hepáticas/tratamento farmacológico , Camundongos Endogâmicos , Apoptose , DinaminasRESUMO
BACKGROUND: Glioblastoma multiforme (GBM), a lethal brain tumor, remains the most daunting challenge in cancer therapy. Overexpression and constitutive activation of PDGFs and PDGFRα are observed in most GBM; however, available inhibitors targeting isolated signaling pathways are minimally effective. Therefore, better understanding of crucial mechanisms underlying GBM is needed for developing more effective targeted therapies. METHODS: Target genes controlled by HIF1α in GBM were identified by analysis of TCGA database and by RNA-sequencing of GBM cells with HIF1α knockout by sgRNA-Cas9 method. Functional roles of HIF1α, PDGFs and PDGFRs were elucidated by loss- or gain-of-function assays or chemical inhibitors, and compared in response to oxygen tension. Pharmacological efficacy and gene expression in mice with intracranial xenografts of primary GBM were analyzed by bioluminescence imaging and immunofluorescence. RESULTS: HIF1α binds the PDGFD proximal promoter and PDGFRA intron enhancers in GBM cells under normoxia or mild-hypoxia to induce their expression and maintain constitutive activation of AKT signaling, which in turn increases HIF1α protein level and activity. Paradoxically, severe hypoxia abrogates PDGFRα expression despite enhancing HIF1α accumulation and corresponding PDGF-D expression. Knockout of HIF1A, PDGFD or PDGFRA in U251 cells inhibits cell growth and invasion in vitro and eradicates tumor growth in vivo. HIF1A knockdown in primary GBM extends survival of xenograft mice, whereas PDGFD overexpression in GL261 shortens survival. HIF1α inhibitor Echinomycin induces GBM cell apoptosis and effectively inhibits growth of GBM in vivo by simultaneously targeting HIF1α-PDGFD/PDGFRα-AKT feedforward pathway. CONCLUSIONS: HIF1α orchestrates expression of PDGF-D and PDGFRα for constitutive activation of AKT pathway and is crucial for GBM malignancy. Therefore, therapies targeting HIF1α should provide an effective treatment for GBM.
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Antibióticos Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/patologia , Equinomicina/uso terapêutico , Glioblastoma/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Linfocinas/metabolismo , Oxigênio/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ativação Enzimática , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Linfocinas/genética , Camundongos , Camundongos Endogâmicos NOD , Invasividade Neoplásica , Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genéticaRESUMO
PURPOSE: To explore whether resveratrol dependents on the production of suppressor of cytokine signaling suppressor 3 (SOCS-3) in inhibiting mRNA production of macrophage inflammatory protein-2 (MIP-2) in osteoblasts induced by lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e). METHODS: MC3T3-E1 cells were treated with different concentrations of resveratrol (0, 5, 10 and 20 µmol/L) and 20 µmol/L resveratrol for different time( 0, 10, 30, 60, 120 and 180 min). The expression of SOCS-3 protein was detected by Western blot. MC3T3-E1 cells were transfected with mouse SOCS3 siRNA (si-SOCS-3) and control siRNA(si-control). Reverse transcription real-time PCR(real-time RT-PCR) and Western blot was used to detect the silencing efficiency of SOCS-3. Cells were stimulated by 20 µg/mL P.e-LPS for 24 h after transfection, in the absence or presence of 20 µmol/L resveratrol for 1 h , and the changes of MIP-2 mRNA were determined by real-time RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS: Treatment of MC3T3-El cells with different concentrations of resveratrol caused a significant increase in SOCS-3 protein expression in a dose-dependent manner. During the observation time of 180 min, SOCS-3 protein expression was the highest at 20 µmol/L resveratrol-treated osteoblasts for 60 min. The silencing efficiency of SOCS-3 mRNA was 63.7%. Transfection with SOCS-3 siRNA increased MIP-2 mRNA expression in LPS-stimulated MC3T3-E1 cells and negated the inhibitory effects of resveratrol on LPS-induced MIP-2 mRNA expression(P<0.05). CONCLUSIONS: Resveratrol inhibits the expression of MIP-2 mRNA in osteoblasts induced by P.e-LPS by up-regulating the expression of SOCS-3 protein.
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Lipopolissacarídeos , Porphyromonas endodontalis , Animais , Lipopolissacarídeos/farmacologia , Camundongos , Osteoblastos , RNA Mensageiro , Resveratrol/farmacologiaRESUMO
Microalgae, as potential biodiesel feedstocks, have been widely reported to accumulate oil via genetic engineering techniques, or environmental stress regulation. Recently, the utilization of fuel cell technology to convert biomass into electricity has attracted much more attention due to its high efficiency, low pollution, low noise by microalgae as feedstocks. Normally, platinum and analogous noble metals as catalysts have been already demonstrated although they still exist lots of shortcomings. This mini review presents an overview of various fuel cell technologies with phosphomolybdic acid as catalysts for sustainable energy by using microalgae. Trends from literatures demonstrate that algal-based fuel cells could efficiently generate electricity, and concurrently produce high value-added products. This critical review can provide guiding suggestions for future study of algal-based energy conversion by fuel cell techniques.
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Hypoxia-inducible factor 1α (HIF-1α) is recognized as a prime molecular target for metastatic cancer. However, no specific HIF-1α inhibitor has been approved for clinical use. Here, we demonstrated that in vivo efficacy of echinomycin in solid tumors with HIF-1α overexpression is formulation-dependent. Compared to previously-used Cremophor-formulated echinomycin, which was toxic and ineffective in clinical trials, liposomal-echinomycin provides significantly more inhibition of primary tumor growth and only liposome-formulated echinomycin can eliminate established triple-negative breast cancer (TNBC) metastases, which are the leading cause of death from breast cancer, as available therapies remain minimally effective at this stage. Pharmacodynamic analyses reveal liposomal-echinomycin more potently inhibits HIF-1α transcriptional activity in primary and metastasized TNBC cells in vivo, the latter of which are HIF-1α enriched. The data suggest that nanoliposomal-echinomycin can provide safe and effective therapeutic HIF-1α inhibition and could represent the most potent HIF-1α inhibitor in prospective trials for metastatic cancer.
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Equinomicina/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Lipossomos/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Equinomicina/química , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Lipossomos/química , Camundongos , Metástase Neoplásica , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: There is conflicting evidence regarding the prognostic value of cytotoxic T cell infiltration in breast cancer. The aims of this study were to detect the expression levels and localization of FoxP3 and CD8 in invasive ductal carcinoma of the breast and to investigate the correlations among FoxP3+ regulatory T cells (Tregs), CD8+ cytotoxic T lymphocytes (CTLs), clinicopathological features, and prognosis in patients with breast cancer. METHODS: Immunohistochemistry was used to detect the expression levels and localization of FoxP3 and CD8. One-sample t-test, one-way analysis of variance, and Kaplan-Meier log-rank tests were used to analyze correlations between the expression levels of CD8 and FoxP3; Kaplan-Meier Log-rank test was used to analyze clinicopathological features to explore the prognostic significance of CD8 and FoxP3 in patients with breast cancer. RESULTS: FoxP3 expression in the tumor bed was higher than that in the stroma, while CD8 was primarily expressed in the stroma. CD8 expression was associated with favorable prognostic factors. However, FoxP3 expression and an increased ratio of total FoxP3+ Tregs to CD8+ CTLs were significantly correlated with unfavorable prognostic factors. Additionally, an increased ratio was associated with molecular subtypes (ER+Her2+, ER+Her2-, ER-Her2+, and ER-Her2-) of breast cancer. Overexpression of FoxP3 and a high FoxP3+/CD8+ ratio were correlated with poor overall survival (OS) and disease-free survival (DFS). However, CD8 expression only affected OS in patients with breast cancer. CONCLUSIONS: Tumor-infiltrating lymphocytes are localized variously depending on the subtype. CD8+ CTLs were associated with a good prognosis, while FoxP3+ Tregs were associated with adverse outcomes in patients with breast cancer. CD8+ CTLs and FoxP3+ Tregs are potential predictive prognostic factors for patients with breast cancer.
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OBJECTIVES: Total flavones from Rhododendron simsii Planch. (TFR) are the effective part extracted from the flowers of Rhododendron simsii Planch. and have obvious protective effects against cerebral ischemic or myocardial injuries in rabbits and rats. However, their mechanism of cardioprotection is still unrevealed. Therefore, the present study was designed to investigate the effect of TFR on myocardial I/R injury and the underlying mechanism. METHODS: TFR groups were treated by gavage once a day for 3 days at a dose of 20, 40, and 80 mg/kg, respectively, and then the model of myocardial I/R injury was established. Myocardial infarction, ST-segment elevation, and the expression of UTR, ROCK1, ROCK2, and p-MLC protein in rat myocardium were determined at 90 min after reperfusion. UTR siRNA in vivo transfection and competition binding assay method were used to study the relationship between the protective effect of TFR and UTR. RESULTS: The expression of UTR protein markedly decreased in myocardium of UTR siRNA transfection group rats. TFR could significantly reduce the infarct size and inhibit the increase of RhoA activity and ROCK1, ROCK2, and p-MLC protein expressions both in WT and UTR knockdown rats. The reducing rate of TFR in myocardial infarction area, RhoA activity, and ROCK1, ROCK2, and p-MLC protein expressions in UTR knockdown rats decreased markedly compared with that in WT rats. In addition, TFR had no obvious effect on the increase of ΣST in UTR knockdown rats in comparison with that in model group. In particular, TFR could significantly inhibit the combination of [125I]-hu-II and UTR, and IC50 was 0.854 mg/l. CONCLUSIONS: The results indicate that the protective effect of TFR on I/R injury may be correlated with its blocking UTR and the subsequent inhibition of RhoA/ROCK signaling pathway.
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TMED2 is involved in morphogenesis of the mouse embryo and placenta. We found that expression of TMED2 was higher in epithelial ovarian cancer tissues than normal ovarian tissues. Silencing TMED2 decreased cell proliferation, migration, and invasion. Ectopic expression of TMED2 increased cell proliferation, migration and invasion. Silencing TMED2 inhibited ovarian cancer growth in mice. Silencing TMED2 inhibited IGF2/IGF1R/PI3K/Akt pathway. In agreement, ectopically expressed TMED2 activated IGF2/IGF1R/PI3K/Akt pathway. Mechanistic study revealed that TMED2 directly binds to AKT2, thereby facilitating its phosphorylation. We also found that TMED2 increased IGF1R expression by competing for miR-30a. Thus, TMED2 is oncogenic and a potential target for epithelial ovarian cancer therapy.
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Hypoxia-inducible factors (HIF) mediate metabolic switches in cells in hypoxic environments, including those in both normal and malignant tissues with limited supplies of oxygen. Paradoxically, recent studies have shown that cancer stem cells (CSCs) and activated immune effector cells exhibit high HIF activity in normoxic environments and that HIF activity is critical in the maintenance of CSCs as well as the differentiation and function of inflammatory cells. Given that inflammation and CSCs are two major barriers to effective cancer therapy, targeting HIF may provide a new approach to developing such treatments.
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Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Epigênese Genética , Humanos , Fator 1 Induzível por Hipóxia/genética , Inflamação/metabolismo , Neoplasias/genética , Neoplasias/patologiaRESUMO
This study examined the effect of Notch-1 signaling on malignant behaviors of breast cancer cells by regulating breast cancer stem cells (BCSCs). BCSCs were enriched by using serum-free medium and knocked out of Notch-1 by using a lentiviral vector. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to detect the Notch-1 expression levels in breast cancer cell lines and BCSCs, and flow cytometry to detect the proportion of BCSCs in BCSC spheres. The BCSC self-renewal, migration, invasion, and tumorigenicity were examined by the tumor microsphere-forming assay and transwell assay and after xenotransplantation. The results showed that the Notch-1 silencing reduced the number of BCSC spheres, the proportion of BCSCs, and the number of cells penetrating through the transwell membrane. It also decreased the size of tumors that were implanted in the nude mice. These results suggest that Notch-1 signaling is intimately linked to the behaviors of BCSCs. Blocking Notch-1 signaling can inhibit the malignant behaviors of BCSCs, which may provide a promising therapeutical approach for breast cancer.
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Neoplasias da Mama/genética , Células-Tronco Neoplásicas/metabolismo , Receptor Notch1/biossíntese , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Receptor Notch1/genética , Transdução de SinaisRESUMO
Lipid droplets (LDs) are a neutral lipid storage organelle that is conserved across almost all species. Many metabolic syndromes are directly linked to the over-storage of neutral lipids in LDs. The study of LDs in Caenorhabditis elegans (C. elegans) has been difficult because of the lack of specific LD marker proteins. Here we report the purification and proteomic analysis of C. elegans lipid droplets for the first time. We identified 306 proteins, 63% of these proteins were previously known to be LD-proteins, suggesting a similarity between mammalian and C. elegans LDs. Using morphological and biochemical analyses, we show that short-chain dehydrogenase, DHS-3 is almost exclusively localized on C. elegans LDs, indicating that it can be used as a LD marker protein in C. elegans. These results will facilitate further mechanistic studies of LDs in this powerful genetic system, C. elegans.
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Biomarcadores/análise , Butiril-CoA Desidrogenase/análise , Proteínas de Caenorhabditis elegans/análise , Caenorhabditis elegans/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteoma/análise , Proteômica/métodos , Animais , Western Blotting , Vesículas Citoplasmáticas/ultraestrutura , Metabolismo dos Lipídeos , Lipídeos/química , Espectrometria de Massas , Microscopia Confocal , Microscopia Eletrônica de TransmissãoRESUMO
Storage of cellular triacylglycerols (TAGs) in lipid droplets (LDs) has been linked to the progression of many metabolic diseases in humans, and to the development of biofuels from plants and microorganisms. However, the biogenesis and dynamics of LDs are poorly understood. Compared with other organisms, bacteria seem to be a better model system for studying LD biology, because they are relatively simple and are highly efficient in converting biomass to TAG. We obtained highly purified LDs from Rhodococcus sp. RHA1, a bacterium that can produce TAG from many carbon sources, and then comprehensively characterized the LD proteome. Of the 228 LD-associated proteins identified, two major proteins, ro02104 and PspA, constituted about 15% of the total LD protein. The structure predicted for ro02104 resembles that of apolipoproteins, the structural proteins of plasma lipoproteins in mammals. Deletion of ro02104 resulted in the formation of supersized LDs, indicating that ro02104 plays a critical role in cellular LD dynamics. The putative α helix of the ro02104 LD-targeting domain (amino acids 83-146) is also similar to that of apolipoproteins. We report the identification of 228 proteins in the proteome of prokaryotic LDs, identify a putative structural protein of this organelle, and suggest that apolipoproteins may have an evolutionarily conserved role in the storage and trafficking of neutral lipids.
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Proteínas de Bactérias/metabolismo , Corpos de Inclusão/metabolismo , Lipídeos/química , Apolipoproteínas/metabolismo , Western Blotting , Cromatografia em Camada Fina , Espectrometria de Massas , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteômica/métodos , Rhodococcus/metabolismo , Triglicerídeos/metabolismoRESUMO
The lipid droplet (LD) is a universal organelle governing the storage and turnover of neutral lipids. Mounting evidence indicates that elevated intramuscular triglyceride (IMTG) in skeletal muscle LDs is closely associated with insulin resistance and Type 2 Diabetes Mellitus (T2DM). Therefore, the identification of the skeletal muscle LD proteome will provide some clues to dissect the mechanism connecting IMTG with T2DM. In the present work, we identified 324 LD-associated proteins in mouse skeletal muscle LDs through mass spectrometry analysis. Besides lipid metabolism and membrane traffic proteins, a remarkable number of mitochondrial proteins were observed in the skeletal muscle LD proteome. Furthermore, imaging by fluorescence microscopy and transmission electronic microscopy (TEM) directly demonstrated that mitochondria closely adhere to LDs in vivo. Moreover, our results revealed for the first time that apolipoprotein A-I (apo A-I), the principal apolipoprotein of high density lipoprotein (HDL) particles, was also localized on skeletal muscle LDs. Further studies verified that apo A-I was expressed endogenously by skeletal muscle cells. In conclusion, we report the protein composition and characterization of skeletal muscle LDs and describe a novel LD-associated protein, apo A-I.